s2fieldsofstudy: Biology Medicine

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Temporal patterns of increased growth hormone secretion in mice after oral administration of l-ornithine: possible involvement of ghrelin receptors

l-Ornithine is known to stimulate growth hormone (GH) release in mammals. Here, we demonstrated that increases in plasma GH levels after oral administration of l-ornithine were first observed 150 min after administration, and the elevated levels were sustained for more than 90 min in mice. The increase was significantly delayed compared with the reported timing of plasma and tissue levels of l-ornithine after administration. The l-ornithine-induced increase in GH release was completely blocked by [D-Lys3]-GHRP-6, a ghrelin receptor antagonist, but not by cyclosomatostatin or JV-1-38, antagonists of somatostatin and GH-releasing hormone, respectively. These results suggest the involvement of ghrelin receptor-mediated pathways in l-ornithine-induced increases in GH release.

ABSTRACT. l-Ornithine is known to stimulate growth hormone (GH) release in mammals. Here, we demonstrated that increases in plasma GH levels after oral administration of l-ornithine were first observed 150 min after administration, and the elevated levels were sustained for more than 90 min in mice. The increase was significantly delayed compared with the reported timing of plasma and tissue levels of l-ornithine after administration. The l-ornithine-induced increase in GH release was completely blocked by [D-Lys 3 ]-GHRP-6, a ghrelin receptor antagonist, but not by cyclosomatostatin or JV-1-38, antagonists of somatostatin and GH-releasing hormone, respectively. These results suggest the involvement of ghrelin receptor-mediated pathways in l-ornithine-induced increases in GH release.
KEYWORDS: ghrelin, growth hormone, growth hormone-releasing hormone, ornithine, somatostatin l-Ornithine is a non-protein amino acid synthesized in the urea cycle and serves as a functional food factor for various physiological functions. For example, l-ornithine intake improves perceived sleep quality and stress marker levels in healthy humans [15]. In rodents, oral administration of l-ornithine increases the duration of non-rapid eye movement sleep [16]; it also modulates the diurnal rhythms of clock gene expression in the peripheral organs [9] and plasma hormonal levels [14]. Several functions may be mediated through the stimulatory role of l-ornithine on growth hormone (GH) release, which is closely linked to sleep physiology.
Previous studies have consistently reported the stimulatory effects of l-ornithine administration on GH release in humans and rodents. However, the temporal patterns of GH response vary with species, protocols, and administration routes. In humans, oral intake of l-ornithine or intravenous infusion after overnight fasting increased serum GH levels 90 and 45 min after administration, respectively [4,7]. A time lag of approximately 45 min occurs in peak levels of serum ornithine and GH after oral administration [4]. On the other hand, in rats administered l-ornithine via the intraduodenal route, plasma GH levels rapidly increased within 15 min and returned to the basal level 60 min after administration [10]. The rapid response observed in rats is mediated through the sympathetic nervous system and ghrelin [10], a stomach-derived hormone that serves as a GH stimulator [12]. In mice, oral administration of l-ornithine increased plasma, hypothalamus, and hypophysis l-ornithine levels within 30-60 min, although it did not acutely increase GH secretion at least 120 min after administration [14]. These reports raise the possibility that the delayed increase in GH release after oral administration of l-ornithine is not directly linked to the distribution of ingested l-ornithine, and the mechanisms might be different from the effect of intraduodenal administration.
Here, we addressed the temporal patterns of GH release during a prolonged period after oral administration of l-ornithine in mice. GH secretion is regulated through a complex neuroendocrine control system that includes ghrelin and two major hypothalamic hormones, somatostatin and GH-releasing hormone (GHRH). Somatostatin is an inhibitor of GH release, secreted from the hypothalamus, gastrointestinal tract, pancreatic islets, and immune cells. GHRH stimulates the transcription of GH mRNA [2] and GH release from somatotrophs. To examine the possible involvement of somatostatin-and GHRH-related pathways, the present study analyzed peripheral levels of somatostatin and hypophyseal GH mRNA levels after l-ornithine administration. Finally, the mediating pathways of oral administration of l-ornithine were investigated using antagonists of ghrelin receptors, GHRH, and somatostatin.
Four-week-old male C57BL/6J mice were purchased from Japan SLC (Shizuoka, Japan) and housed in plastic cages in groups of three or four mice. We used young C57BL/6J mice because their GH release is highly sensitive to the oral administration of l-ornithine, as we observed in our pilot experiments. In all experiments, the cages were placed in a room at 25 ± 1°C ambient temperature, under light conditions of 12 L:12D, for at least one week before the experiments began. Water and a standard diet for laboratory rodents (MF, Oriental Yeast; Tokyo, Japan) were provided ad libitum. The study was conducted according to the Guidelines for Animal Experiments of the Faculty of Agriculture at Kyushu University, following Law No. 105 and Notification No. 6 of the Japanese Government. All experiments were approved by the Animal Care and Use Committee of Kyushu University (A30-022-0).
Mice were administered either distilled water (10 mL/kg) or l-ornithine monohydrochloride (3 g/kg, dissolved in distilled water, 10 mL/kg) by oral gavage at Zeitgeber time (ZT: ZT0 represents light onset) 8. The timing was selected to avoid internal increases in GH release during ZT3-4. Mice were fasted from ZT14 on the previous day to avoid an acute dietary effect (18 hr fasting in total). The mice were euthanized at 0, 30, 60, 90, 120, 150, 180, and 240 min after administration using isoflurane gas and decapitated. After decapitation, trunk blood was collected in heparinized tubes and centrifuged at 3,000 × g for 10 min at 4°C to obtain plasma samples for GH assays. The hypophysis was harvested for assays at 0, 150, 180, and 240 min and stored in RNAlater (Thermo Fischer Scientific; Waltham, MA, USA) for quantitative PCR (qPCR). Plasma GH concentrations were measured in duplicate using a Rat/ Mouse GH ELISA kit (EMD Millipore, Billerica, MA, USA) according to the manufacturer's protocol. The detection limit for the assay was 0.07 ng/mL. The intra-and inter-assay coefficients of variation were 2.6 and 4.2%, respectively.
Another cohort of mice was administered either distilled water or l-ornithine monohydrochloride as described above. The mice were euthanized at 0, 120, 180, and 240 min following oral administration using isoflurane gas and decapitated. The corpus of the stomach and duodenum were dissected and stored in RNAlater (Thermo Fischer Scientific) for qPCR.
We used cyclosomatostatin (cSST), a somatostatin antagonist, [D-Lys 3 ]-GHRP-6 (DLys), a ghrelin receptor antagonist, and JV-1-38, a GHRH antagonist, to show the involvement of GH regulatory systems (somatostatin, ghrelin, and GHRH) on l-ornithine-induced increases in GH release. cSST and DLys were obtained from Sigma-Aldrich (St. Louis, MO, USA), and JV-1-38 was obtained from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA). cSST and DLys were dissolved in saline, while JV-1-38 was dissolved in 0.1% dimethyl sulfoxide in 10% aqueous propylene glycol solution (DMSO-PG). The mice were fasted from ZT14 on the previous day. On the day of the experiment, the mice were intraperitoneally injected with saline, DMSO-PG, cSST (12 μg/kg) [8], DLys (5.5 mg/kg) [1], or JV-1-38 (100 μg/kg) [21] at ZT8. Five min after injection, water or l-ornithine monohydrochloride (3 g/kg) was orally administered. The mice were euthanized at 180 min following oral administration using isoflurane gas and decapitated. After decapitation, plasma samples were obtained as described above. The antagonists were injected prior to l-ornithine administration because we aimed to inhibit the effects of l-ornithine or metabolites on these pathways during the 180 min interval following oral administration.
Because the inhibitory effect of JV-1-38 on GH response to GHRH was less than 40% at 60 min in rats [21], another batch of mice was injected with JV-1-38 or DMSO-PG three times at 0 (5 min before l-ornithine administration), 60, and 120 min. The inhibitory effects of cSST and DLys on physiological parameters, i.e., haloperidol-induced catalepsy in rats and food intake in food-deprived mice, respectively, lasted for at least 4 hr [1,11].
Total RNA was extracted from the hypophysis, stomach, and duodenum using RNAiso Plus (Takara, Kusatsu, Japan) according to the manufacturer's protocol. cDNA was synthesized using 1 µg of total RNA and PrimeScript RT Reagent Kit with gDNA Eraser (Takara). qPCR was performed using Stratagene Mx3000P (Agilent Technologies; Santa Clara, CA, USA). Each mRNA level was calculated using threshold cycles for the amplification of unknown samples and compared with those of six concentrations of the standards. The calculated levels were normalized to the mRNA levels of hypoxanthine phosphoribosyltransferase (HPRT). The primer sequences were as follows: GH forward 5′-GCTACAGACTCTCGGACCTC-3′, reverse 5′-CGGAGCACAGCATTAGAAAACAG-3′; ghrelin forward 5′-TCCAAGAAGCCACCAGCTAA-3′, reverse 5′-AACATCGAAGGGAGCATTGA-3′; HPRT forward 5′-ATACAGGCCAGACTTTGTTGGATT-3′, reverse 5′-TCACTAATGACACAAACGTGATTCAA-3′. Melting curve analysis was performed for each gene to confirm the specificity of the PCR conditions.
Temporal patterns of plasma GH or somatostatin levels, as well as hypophyseal GH mRNA levels, after l-ornithine administration were analyzed by two-way ANOVA followed by Bonferroni's multiple comparison test between water-and l-ornithine-administered mice at each time point. Equal variances were confirmed using the Brown-Forsythe test. At each time point, these levels were also compared to the initial levels (0 min before administration) by Dunnett's test. In antagonist experiments, the effects of saline-dissolved compounds (cSST and DLys) and the DMSO-PG-dissolved compound (JV-1-38) were analyzed separately. Because variances differed between groups, we used the Kruskal-Wallis test followed by Dunn's multiple comparison test using vehicle-and water-treated mice as controls.
When the plasma GH levels were compared between water-administered mice and l-ornithine-administered mice at each time point, significant increases in plasma GH levels started appearing 150 min after administration (P=0.0083, 0.0003, and 0.0026 at 150, 180, and 240 min, respectively) (Fig. 1A). The increased levels were sustained until at least 240 min after administration (Fig.  1A). Similarly, comparative analysis with initial levels revealed significant increases in plasma GH levels at 150, 180, and 240 min (P=0.0023 for 150 min, P<0.0001 for 180 and 240 min). In the water-administered mice, endogenous increases in plasma GH levels were observed at 180 and 240 min (P=0.0475 and 0.0001, respectively) (Fig. 1A). Hypophyseal GH mRNA expression was not modified by l-ornithine administration when plasma GH exhibited remarkable increases (150-240 min) (Fig. 1B).
Plasma somatostatin levels transiently increased 30 min after l-ornithine administration (P=0.0041), but minimal effects were observed after 60 min (Fig. 1C). A negative correlation was observed between plasma GH and somatostatin levels in l-ornithineadministered mice (r= −0.4269, P=0.0186) (Fig. 1D). Ghrelin mRNA levels in the stomach and duodenum were not significantly altered after l-ornithine administration (Fig. 1E, F). The levels at 120, 180, and 240 min after oral administration did not significantly differ from the initial levels observed in l-ornithine-administered and water-administered mice.
In antagonist experiments, l-ornithine-administered mice with a prior injection of saline exhibited significant increases in plasma GH levels 180 min after administration (P=0.0341) (Fig. 2A). This increase was not completely inhibited by prior injection of cSST (P=0.0537). In contrast, injection of DLys completely blocked this upregulation ( Fig. 2A). In mice injected with DMSO-PG, l-ornithine administration increased plasma GH levels (P=0.0368). JV-1-38 did not completely block this response, and a high individual variation was observed (Fig. 2B). Because the variation might be derived from the short-term efficiency of JV-1-38, we repeated injections three times every 60 min. Although DMSO-PG injections diminished the l-ornithine-induced increase in GH levels (P=0.1101), JV-1-38 injected mice exhibited significantly higher levels of plasma GH than the control (P=0.0107) (Fig. 2C).
The present study showed that the onset of increases in GH release in mice by oral administration of l-ornithine was delayed up to 150 min after administration. This timing was relatively slow compared with plasma, hypothalamus, and hypophysis levels of l-ornithine in mice, which were reported to have peaked at 30 min, followed by a rapid decrease up to 120 min after administration [14]. We showed that the delayed increase was mediated through post-transcriptional pathways and was completely blocked by 1283-1287, 2022 Temporal changes in plasma GH levels (A), hypophyseal GH mRNA levels (B), and plasma somatostatin levels (C) after water or l-ornithine (3 g/kg, l-ornithine monohydrochloride) administration. **P<0.01, ***P<0.001, Bonferroni's multiple comparison test. Different characters indicate significant differences compared with the initial levels (before administration) by Dunnett's test. Values are mean ± SEM (n=4-6).
(D) Correlation between plasma GH and somatostatin levels in l-ornithine-administered mice. (E, F) Temporal changes in ghrelin mRNA levels in the stomach and the duodenum after water or l-ornithine administration.
prior injections of antagonists of ghrelin receptors. These data suggest that the mechanisms include indirect effects of l-ornithine on GH release through ghrelin receptors. Somatostatin and GHRH antagonists did not completely block the l-ornithine-induced increase in GH release. This might be due to high individual effect variations or efficiency persistence. More powerful loss-of-function methods, such as knockout studies, need to be conducted in the future.
Our study showed that plasma somatostatin levels transiently increased after oral administration of l-ornithine, which was negatively correlated with plasma GH levels. However, the correlation was slightly weak, and plasma GH levels did not significantly differ at increased time intervals (30 min). Thus, the impact of the transient increases on short-term GH regulation remains unclear. Mechanisms for the early stimulation of somatostatin release may include glucagon-like peptide 1 (GLP-1), which is strongly induced by l-ornithine in cultured GLUTag cells [18] and mice 15 min after oral administration [5]. GLP-1 infusion increased somatostatin secretion in the perfused pancreas of rats [6] and mice [17]. As somatostatin has multiple functions, including antiproliferative effects and inhibition of trophic hormones, such as insulin-like growth factor and epidermal growth factor [3], the transient increase may have other physiological effects associated with these functions.
Ghrelin is a gut-derived hormone isolated as an endogenous ligand for GHS-R and identified as a potent GH stimulator [12]. The stimulatory effects are elicited directly or indirectly through GHS-R localized in the hypophysis and hypothalamus, respectively. Thus, l-ornithine-induced increases in GH release may include the stimulation of ghrelin production in the gut. This hypothesis is supported by a previous study showing that intraduodenal administration of l-ornithine upregulated ghrelin expression in the duodenum 30 min after administration in rats [10]. However, in the present study, oral administration of l-ornithine did not alter ghrelin expression in the stomach and the duodenum 120-240 min after administration in mice, the interval wherein GH release was increased. Although we could not measure n-octanoyl circulatory ghrelin concentrations, the results suggest that l-ornithine-induced increases in GH release may not involve the long-term stimulation of peripheral ghrelin production.
Alternate mechanisms may involve the modulation of GHS-R activity by l-ornithine. GHS-R is localized to the hypophysis, where ghrelin directly stimulates GH release [20]. GHS-R is a G proteincoupled receptor (GPCR) that can cross-talk with other GPCRs to modulate receptor sensitivity [19]. Considering that one of the GPCRs (GPRC6A) is an l-ornithine sensor [18], l-ornithine may modulate GHS-R sensitivity to ghrelin by interacting with other GPCRs. In this hypothesis, GHS-R activation is necessary to elicit enhanced GH release. Notably, in the present study, l-ornithineinduced GH release started to increase along with endogenous increases in GH release. The endogenous changes might be due to prolonged fasting because the mice were fasted for 18 hr before experiments. In a previous study, prolonged fasting (24 hr and 48 hr before experiments) is associated with increased GHS-R and ghrelin expression in the murine hypophysis [13]. These data suggest that GH response to ghrelin or ghrelin production in the hypophysis might increase during prolonged fasting, and l-ornithine administration might enhance the response.
In conclusion, oral administration of l-ornithine increased GH release in mice, and the regulatory pathway is likely to involve 1283-1287, 2022 ghrelin receptors. The onset of GH increases was delayed up to 150 min after oral administration, suggesting a sensitizing effect on ghrelin receptors in hypophyseal GH cells. Our study suggests a novel mechanism of l-ornithine action as a hormonal regulator. The cooperative mechanisms of l-ornithine and ghrelin receptors need to be further investigated in future studies.


Genetic code degeneracy is established by the decoding center of the ribosome

Abstract The degeneracy of the genetic code confers a wide array of properties to coding sequences. Yet, its origin is still unclear. A structural analysis has shown that the stability of the Watson–Crick base pair at the second position of the anticodon–codon interaction is a critical parameter controlling the extent of non-specific pairings accepted at the third position by the ribosome, a flexibility at the root of degeneracy. Based on recent cryo-EM analyses, the present work shows that residue A1493 of the decoding center provides a significant contribution to the stability of this base pair, revealing that the ribosome is directly involved in the establishment of degeneracy. Building on existing evolutionary models, we show the evidence that the early appearance of A1493 and A1492 established the basis of degeneracy when an elementary kinetic scheme of translation was prevailing. Logical considerations on the expansion of this kinetic scheme indicate that the acquisition of the peptidyl transferase center was the next major evolutionary step, while the induced-fit mechanism, that enables a sharp selection of the tRNAs, necessarily arose later when G530 was acquired by the decoding center.

INTRODUCTION
The translation of genetic information relies on base pairing between anticodons and codons. While the first two codon positions are restricted to canonical Watson-Crick base pairs, some flexibility occurs at the third position. This flexibility was postulated by F. Crick in 1966 to account for the observed degeneracy in the genetic code, which had just been fully deciphered (1). He suggested that G could base pair not only with C but also with U if some base displacement was possible at the third position, a possibility coined the 'wobble hypothesis' (2). This flexibility would allow reduced sets of tRNAs to translate all amino-acid en-coding codons, thereby making translation more efficient. The reason why unspecific pairing can be accepted at the third position became apparent only about 35 years later when the first structures at atomic resolution of the 30S subunit co-crystallized with mRNA fragments and anticodon stem-loops were elucidated (3). These structures revealed that, unlike at the first and second position, the ribosome does not structurally constrains the wobble position, implying that some flexibility in the geometry of base pairing is possible (3)(4)(5).
In the meantime, it was discovered that extended wobbling, called 'superwobbling' (6), can also occur at the third position. In that case, an unmodified U (exceptionally an A) at position 34 of a tRNA can base pair with any base at the third position of the codons (7). So far, superwobbling has been observed only in mitochondria, chloroplasts and other small genome entities with reduced sets of tRNAs (7)(8)(9). In such cases, the extent of wobbling matches the degeneracy families associated with each of the 16 N 1 N 2 codon doublets of the genetic code: all codons of any codon family, whether it is two-or four-fold degenerate, are translated by a single tRNA through wobbling and superwobbling, respectively.
The rationale behind the existence of these two degeneracy families was partially unraveled in 1978 by U. Lagerkvist, who noticed that the strength of the base pairs in positions 1 and 2 of the codons and the purine/pyrimidine nature of the base at the second position constituted a set of three criteria (or parameters) with which a complete categorization of the 16 codon doublets into the two degeneracy families was possible (10), a feature that can be highlighted by a symmetry in the genetic code table (11). Based on the then available structural organization of the decoding center, and the architecture of the anticodon loop, an interpretation of these parameters was proposed in 2008. The analysis (12) demonstrated that all three parameters of Lagerkvist determine the number of hydrogen bonds contributing to the stability of the WC geometry of the base pair at the second position of the anticodon (N 35  Considering the sum S of hydrogen bonds defined by the three parameters, it was shown that when S ≤ 5, the considered codon belongs to a two-fold degenerate family, while it belongs to a four-fold degenerate family if S > 5 (12). The WC geometry of N 35 -N 2 is critical since it is closely monitored by A1492 and G530 of the decoding center. These residues control 30S closure (3,4) through an induced-fit mechanism which triggers GTP hydrolysis on EF-Tu and the subsequent release of the tRNA for accommodation (14)(15)(16). This geometry can be perturbed by non-WC base pairs at the third position of the codons. The model shows that penalizing N 34 -N 3 mismatches can sufficiently alter that geometry to prevent the decoding center from adopting a productive configuration. With S > 5, any perturbation by the four possible base pairs between (usually) U 34 and N 3 is contained by N 35 -N 2 , and superwobbling is possible, whereas base pairing is restricted to simple wobbling when S ≤ 5, which has allowed the encoding of two different amino acids (or an amino acid and the stop function) by the considered N 1 N 2 doublet during the expansion of the initial genetic code.
At the time when this model was published, the dynamics of the decoding center was unknown, and its three residues (A1493, A1492 and G530) were assumed to be either all in the OFF or all in the ON state (resp. syn and anti for G530), the latter case corresponding to a situation where they are tightly packed and form hydrogen bonds along the minor groove of the anticodon-codon complex. In that state, the ribosome is engaged to accept the tRNA (3,4,14,17). This a priori type of dynamics implied that an essential aspect of the model was unsatisfactory: in the all-OFF state, the respective contributions of the hydrogen bonds of N 35 -N 2 and N 36 -N 1 to the stability of the N 35 -N 2 base pair were identical (as it reflects in S), which was physically implausible. To resolve this inconsistency, it was envisioned (although not clearly stated) that residue A1493 would always be in the ON state when N 36 -N 1 and N 35 -N 2 were complementary, even in the occurrence of penalizing mismatch at the third position. In that state, this residue binds to N 36 -N 1 through a type I A minor interaction, which is stronger with G 36 -C 1 and C 36 -G 1 as compared to A 36 -U 1 and U 36 -A 1 , thereby amplifying the difference already present between these pairs. A structural context with N 36 -N 1 as a triple base pair (N 36 -N 1 -A 1493 ) would explain why N 35 -N 2 and N 36 -N 1 had an apparent similar weight in the stability of the N 35 -N 2 base pair. It implied, however, that the decoding center would be already partially ON even though the tRNA could still be rejected by the ribosome. Insights into the dynamics of the decoding center were necessary to clarify this issue.
Two recent cryo-EM analyses (15,18) were able to highlight details of this dynamics, which allow us now to confirm the anticipated role of A1493 in degeneracy and reveal that the degeneracy of the genetic code results not only from reduced structural constraints at the wobble position but also from the participation of A1493 in the stability of the N 35 -N 2 base pair. These new results clearly show that the whole organization of the genetic code is intimately connected to the ribosome itself.
In the first part of the paper, an improved structural model of degeneracy is outlined in the light of the cryo-EM analyses of Loveland et al. (15) and Fislage et al. (18).
The second part relates the dynamics of the decoding center outlined in the first part to the evolution of decoding on the ribosome, an analysis underpinned by models of ribosome evolution (19,20). We show that this dynamics is consistent with an early appearance of A1493 and A1492 on helix h44, at a time when no catalytic site was present and when an early kinetic scheme of translation that did not include tRNA accommodation was prevailing. In this early kinetic scheme, inferred from a physicochemical correlation in the genetic code, our analysis suggests that the initial role of A1493 and A1492 was to allow a relaxation of base pairing specificity at the third position of the codons through the compensatory strengthening they implemented at the first position, which gave rise to degeneracy. Kinetics considerations suggest that the peptidyl transferase center (PTC) was the next major acquisition by the ribosome, while proofreading (21)(22)(23) arose at a later stage together with tRNA accommodation and the initial form of EF-Tu•GTP. Our analysis leads us to conclude that the controlled hydrolysis of EF-Tu' GTP through 30S closure by induced fit was a latecomer mechanism, implemented when G530 was acquired by the decoding center.

Analysis of ribosome structures
Crystal and cryo-EM structures of ribosomes complexed with tRNAs or tRNA fragments (15,18,24) were retrieved from the protein databank website and analyzed with the Pymol software.  (18)). Although A1493 is ON, no hydrogen bond with the minor groove can occur. CryoEM resolution is 3.4Å. (B) Non-cognate interaction, with GU mismatch at the second position in the state of tRNA sampling (pdb 5uyp, (15)). A1493 binds to the minor groove. Hydrogen bond D-A lengths are 1: 3.6Å; 2: 3.0Å; 3: 4.5Å (avg.: 3.7Å). CryoEM resolution is 3.9Å. (C) Cognate interaction in the state of tRNA sampling (pdb 5uyl, (15)). A1493 binds to the minor groove. Hydrogen bond D-A lengths are 1: 3.0Å; 2: 3.1Å; 3: 3.8Å (avg.: 3.3Å). CryoEM resolution is 3.6Å. (D) X-ray structure of a cognate interaction (pdb 1xnq, Murphy and Ramakrishnan 2004) illustrating an A minor interaction with a GC base pair at the first position. Hydrogen bond D-A lengths are 1: 2.6Å; 1': 2.9 A; 2: 3.3Å; 3: 2.5Å (avg.: 2.8Å). Compared to pdb 5uyl, examination of the 5uym pdb structure suggests that the shorter length of these bonds results from A1493 and A1492 being both bound to the anticodon-codon complex. X-ray resolution is 3.05Å. In order to highlight hydrogen bonds, the angle of view was tilted compared to the other structures, and A1492 is semi-transparent. Overall, A1492 is found about 50% of the time in the 'ON' state during tRNA sampling (18). Specific densities of A1492 are such that it is 50% ON/50% OFF in the 5wfk structure (light pink), ON in the 5uyp structure (red) and OFF in the 5uyl structure (red).

PART I : Degeneracy on modern ribosomes
The structural model of degeneracy is consistent with cryo-EM data. Recent cryo-EM investigations on the decoding mechanism of the ribosome have allowed the identification of three different states of the decoding center and the A-site tRNA in the timeline from initial tRNA ribosome binding down to 30S closure (15,18). Following Fislage et al.'s notations (18) (see Figure 7 of their publication), these states are: initial tRNA binding, tRNA sampling and engaged state, the latter state corresponding to a closed 30S subunit, in which the ribosome commits to accept a tRNA. Although the temporal order of these states could be questioned (a common issue in cryo-EM), this order is consistent with our own analysis, as outlined below.
The structures show that with a single mismatch at either the first or the second position of the codon, or in the cognate case, residue A1493 moves to and remains in the 'ON' position during tRNA sampling, i.e. flipped out of helix 44 and in N 36 -N 1 minor groove binding position.
With a A 36 -C 1 mismatch at the first position, A1493 does not form hydrogen bonds with the minor groove, no AC pair being formed (Figure 2A). With a G 35 -U 2 mismatch at the second position, a A 36 -U 1 base pair does form, and A1493 binds to its minor groove, although none of its three hydrogen bonds is optimal ( Figure 2B). In the cognate case, A1493 binds to the minor groove and forms h-bonds during tRNA sampling ( Figure 2C).
There is no existing structure with a forbidden base pair at the third position only, for which the model predicts that A1493 would, likewise, bind to the first base pair during tRNA sampling. The above data, however, clearly support this possibility.
Because the wobble position is two base pairs away from the A1493 binding site, a N 34 -N 3 mismatch generates a smaller perturbation at the A1493 binding site than a N 35 -N 2 mismatch, for which A1493 A minor binding during tRNA sampling is now confirmed ( Figure 2B). A complete demonstration would, however, require a structure with a base pair more penalizing than G 34 -U 3 at the third position, e.g. U 34 -U 3 or U 34 -C 3 (U 34 is almost always involved in superwobbling, see (7,9)).
In brief, cryo-EM analyses have revealed that the A1493 residue of the decoding center binds to the minor groove of N 36 -N 1 during tRNA sampling if this base pair is Watson-Crick, a binding that further stabilizes the complex during the time it is tested by residues A1492 and G530 for 30S closure.
Degeneracy in the genetic code is established through a major contribution by A1493. The cryo-EM data of Loveland et al. and Fislage et al. (15,18) allow us to refine the structural model of degeneracy previously described (12). Figure  3A highlights the four different levels specifying the stability of the WC geometry of the N 35 -N 2 base pair during tRNA sampling in the situation when both N 36 -N 1 and N 35 -N 2 are complementary. The two lowest levels attribute a twofold degeneracy to the corresponding codons, while the two highest levels attribute a four-fold degeneracy. As a result of the equivalence of Lagerkvist's parameters, levels 2 and 3 are degenerate in such a way that three configurations of hydrogen bonding patterns are possible. Remarkably, to each configuration correspond two sets of codons related by A 1 ↔ U 1 or G 1 ↔ C 1 permutations (indicated on each anticodon stem in Figure 3A). Consequently, when A 1 (G 1 ) and U 1 (C 1 ) are mirror ordered with respect to the center of the table (dashed line), the two degeneracy families are also symmetrically arranged with respect to the center ( Figure  3B).
According to the analysis, the most remarkable effect that occurs when both N 36 -N 1 and N 35 -N 2 are complementary is the positive selection of tRNAs enforced by A1493: the strengthening of N 35 -N 2 resulting from N 36 -N 1 A minor binding enables the acceptance of some tRNAs with non-WC base pairs at the third position, whereas tRNAs are counterselected when N 36 -N 1 and/or N 35 -N 2 are not complementary (3,4,15,18).
The involvement of A1493 in degeneracy provides an explanation for why Lagerkvist's parameters are equivalents ( Figure 3A): each increase in the level of stability of N 35 -N 2 occurs upon the addition of either 1 local hydrogen bond (U 33 -N 35 or N 35 -N 2 , in blue) or 2 hydrogen bonds on the neighboring triple base pair (N 36 -N 1 -A 1493 , one in blue and one in red), revealing that these two possibilities are equivalent in term of the added stability to N 35 -N 2 .
A striking aspect of the model is that no stacking parameter is required. It suggests that the high number of hydrogen bonds involved (7 to 11) confer structural energies that dominate over the variability of the stacking interaction, which further corroborates the implication of A1493 in degeneracy. The number of these hydrogen bonds is invariant upon A 1 ↔ U 1 or G 1 ↔ C 1 permutations. In the case of G 36 -C 1 and C 36 -G 1 , this property stems from the position of the G 1/36 (C 1 -NH 2 ) amino group at the center of the base pair ( Figures 2D and 3B). While the length of these bonds may vary (and may not be accurately determined due to structure resolution), they can all be categorized as 'strong': during tRNA sampling, the average DA distance is 3.3Å for the hydrogen bonds of the A minor between A1493 and the U 1 -A 36 base pair and 3.15Å for the hydrogen bonds involved in U 1 -A 36 and U 2 -A 35 base pairing ( Figure 2C).
Although stacking is not a parameter, N 37 stabilizes the N 36 -N 1 base pair by stacking on it, an effect that is optimal since this base is a conserved purine (13). Stabilization is further enhanced when N 37 is modified (25)(26)(27)(28)(29), and the extent of modification negatively correlates with the G + C composition of the anticodon (25,28), indicating that this base also contributes to an adjustment of the overall stability of each anticodon-codon interaction, and is thus possibly a hidden requirement to the observed degeneracy. Deformation of the tRNA body has also been shown to affect the extent of wobbling at the third position (see Summary and Discussion section).
With regard to the present analysis, the directional nature of hydrogen bonds, which is more restrictive compared to that of stacking, and is thus is more prone to keep residues in a given orientation, plausibly explains why they play a predominant role in the stability of the geometry of the N 35 -N 2 WC base pair, which is the decisive criteria for ribosome closure (15,18). In a situation relevant to degeneracy (Figure 3A), this geometry is preserved if the network of hydrogen bonds stabilizing N 35 -N 2 is strong enough to contain the perturbation generated by a given non-canonical N 34 -N 3 base pair.

PART II : Evolution of decoding on the ribosome
The induced-fit mechanism is a late acquisition of the decoding center. The implication of A1493 in degeneracy shows that the implementation of unspecific pairing at the third position of the codons arose at the time when the ribosome acquired residue A1493 on helix h44. Remarkably, two analyses suggest that the segment of h44 where A1493 and A1492 are located appeared early in the evolution of the ribosome, whereas helix h18, harboring G530, emerged at a much later stage (19,20) ( Figure 4A). The latter residue has a major role in the induced-fit mechanism: it drives 30S closure (15,18), which triggers GTP hydrolysis on EF-Tu, thereby releasing the incoming tRNA for accommodation (14,16,30). The mentioned models on ribosome evolution are thus consistent with the induced fit of the decoding center being, logically, established later than degeneracy.
The connection between the successive emergence of helices h44 and h18 and these fundamental aspects of translation must be underscored. The mechanism itself reflects this evolutionary succession: A1493 first binds to the mi-Nucleic Acids Research, 2022, Vol. 50, No. 7 4117  . Amino acids are not specified to point out that they are not primarily involved in the determination of these families. The A minor interaction between A1493 and N 36 -N 1 is shown on the left. All shown hydrogen bonding patterns were found in experimental structures (see Figure 2), except that of C 36 -G 1 -A 1493 , for which no structure could be identified in the pdb database. In that case, the only hypothetical hydrogen bond, highlighted with an asterisk*, is expected to occur similarly as for the G 36 -C 1 -A 1493 configuration due to the position of the G 1/36 (C 1 -NH 2 ) amino group at the center of the base pair. Michaelis-Menten type of kinetic inferred from the volume correlation (11) governs the rate of translation, with tRNA association (k + ) and dissociation (k-) rate constants, and a kinetic constant of peptide bond formation (k pep ), sometimes called k cat in earlier works (11,31,32). The advent of U 33 and R 37 , as well as helix h44 (A1493 and A1492) modulated these kinetic constants (k' + , k'-). The step of tRNA accommodation, that appeared concomitantly with EF-Tu, is characterized by an approximately uniform kinetic constant k acc . Relevant features are shown above each evolutionary stage: (0) Volume correlation. The anticodon-codon G 0 s are assumed to relate to dissociation rate constants k-s through k-∼ exp ( G 0 ) (11), while data on intramolecular reactions suggest a similar exponential dependence between k pep and van der Waals volume (33,34), with some apparent exceptions (asn, arg, trp). nor groove, while A1492 fluctuates between ON and OFF states; only then can A1492 and G530 fully bind to the complex in the cognate case, thereby achieving ribosome closure (15,18).
A model of the early translation before the appearance of helix h44. The involvement of A1493 in degeneracy highlighted by the present analysis, and the coherence of the sequential buildup of the decoding center in the evolutionary models of Harish and Caetano-Anollés (19) and Petrov et al. (20) motivated us to outline a model of evolution of ribosomal decoding based on the identified role of A1493 and a plausible form of the earliest kinetic scheme of translation (32).
This kinetic scheme ( Figure 4B, left) was established from an interpretation of a physico-chemical correlation in the genetic code called the volume correlation (11,31,35). This correlation suggests that at the origin of translation, the lifetime of the association between a tRNA and a complementary codon was about equal to the characteristic time required by the aminoacyl carried by this tRNA to make a peptide bond, which was strongly side-chain dependent. This adjustment, which can be expressed with kinetic constants as kanticodon-codon ≈ k pepaminoacyl (in short: k-≈ k pep ), implies that the aminoacyls were in immediate position for forming a peptide bond upon tRNA codon binding--i.e. there was no tRNA accommodation at the origin--while not being confined inside a catalytic site, which would have standardized the k pep s to an approximately uniform value, an action that is achieved by the peptidyl transferase center (PTC) of modern ribosomes (35). An elementary Michaelis-Menten kinetic scheme comprising the above kinetic constants best encapsulates these features ( Figure 4B, left).
In this kinetic scheme, the dissociation rate constant of the tRNA on the P site after peptide bond formation is naturally assumed to be equal to the k-on the A site, a symmetry that explains why, in the early kinetic scheme, the rate of translation is optimal precisely when k-≈ k pep occurs for all tRNA:aminoacyl couples (Cibils et al., in prep.). Key aspects that account for this property are briefly described below.
In order to ensure peptide bond formation, it seems the stronger the anticodon-codon interaction the better: hypothetical anticodons made up of a large number of nucleotides (low k-) would make a tRNA stay a long time on a cognate codon (much longer than the time required by the reaction) and peptide bond formation would always occur. However, once peptide bond formation has occurred, a high rate of translation requires that the (previous) tRNA dissociates as fast as possible (high k-) so that translocation can take place to let the subsequent codon available for a next incoming tRNA.
On the whole, there is an optimum in the lifetime of the anticodon-codon association that ensures (i) a reasonably good probability of peptide bond formation p = k pep / (k-+ k pep ), essentially half of the time according to the above equality, and (ii) a fast dissociation of the tRNA that has done its job and must leave the complex. The volume correlation suggests that most tRNA:aminoacyl couples follow the k-≈ k pep equality enabling a high translation rate, which also provides a rational for the size of three nucleotides the (anti)codons.
Another line of analysis suggests that the volume correlation may, alternatively, reflect an optimization in decoding fidelity. In this regime, k-is slightly smaller than k pep , which ensures the best discrimination of complementary interactions from noncomplementary ones, a property that directly results from the theory of optimal decoding developed by Y. Savir and T. Tlusty (36,37). The volume correlation could either reflect an optimization in decoding or in the rate of translation, or a solution in between these two regimes, that are very close.
The appearance of helix 44 with A1493 and A1492 generated a decoding transition on the ribosome. In the context defined by the model of the early translation outlined in the previous section, a straightforward consequence of the strengthening of the N 36 -N 1 base pair that occurred when A1493 became functional on h44 was a relaxation of base pairing specificity at the third position of the codons, a rebalancing scheme that would have overall preserved the k-, and thus the rate of translation. In a context of a limited variety of tRNAs, this action of A1493 presumably led to an increase in the processivity and accuracy of translation, discussed below.
Because a mismatch perturbs the geometry and stability of neighboring base pairs along a double helix, the type I A minor binding achieved by A1493 could have been optimal only at the first position, i.e. two base pairs away from the third position (where tolerated mismatches would occur), which may explain why this solution was selected.
Although models of ribosome evolution may not predict whether A1493 and A1492 were both initially present on h44 (19,20), this possibility is plausible since the dynamics of A1493 would likely be altered without A1492, and the type II A minor binding achieved by A1492 (3), which is more tolerant to mismatch (it does not bridge over N 35 -N 2 ), may contribute to N 36 -N 1 stabilization. This binding occurs ∼50% of the time during tRNA sampling (18). In that state, the tRNA is partially bent, a feature associated with presence of EF-Tu that allows an optimal substrate selection through deformation (17,(36)(37)(38)(39). Because EF-Tu, an elaborate protein cofactor, could not have occurred at the origin of translation (which is consistent with an absence of tRNA accommodation, inferred from the volume correlation), it can be affirmed that the stem of initial tRNA adapters did not undergo such deformation. In that case, A1492 would bind 100% of the time to the complex upon tRNA codon association, similarly as it does with fully accommodated tRNAs on modern ribosomes. A fully bound A1492 may contribute to an optimal strengthening mediated by A1493 in the situation when both N 36 -N 1 and N 35 -N 2 are Watson-Crick.
Structural and functional considerations suggest that both the processivity and accuracy of translation increased when residues A1493 and A1492 became functional on helix h44: Processivity of translation: In the proposed Michaelis-Menten kinetic scheme of the initial translation ( Figure 4B), the relaxation in base pairing specificity that occurred at the third position of the codons through the action of A1493 and A1492 may have allowed a given set of different transfer tRNAs, necessarily limited at the origin, to be more tolerant to mutations incorporated at the third position during replication ( Figure 5), and thus translate longer sequences.
As there was initially no strong geometrical requirement for the base pair at the second position in the absence of G530 and induced-fit mechanism, unspecific base pairing at the third position, that perturb the N 35 -N 2 geometry, was plausibly less stringent than that occurring on modern ribosomes.
A1493 and A1492 binding would compensate for the loss in anticodon-codon stability generated by mismatches at the third position within a simple rebalancing scheme (Figure 5A, Table 1). It suggests to us that the four codons belonging to any of the 16 N 1 N 2 doublets of the genetic code may have been translated by a single tRNA upon the action of h44 in an all four-fold-degeneracy regime following transition 1 (Figure 6, center). This possibility does naturally not imply that all 16 doublets were encoding amino acids, at least immediately following this early transition. The acquisition of R 37 and U 33 on the anticodon loop, that presumably also occurred early in the evolution of the tRNAs, respectively reinforced the N 36 -N 1 base pair through R 37 stacking and provided an extended conformational freedom to N 34 at the edge of the U-turn (40), in an apparent synergism with the effect of h44 ( Figure  5A). The early replication mechanism being inaccurate, the arising degeneracy most likely improved the processivity of translation among mutated copies of early RNA genes ( Figure 5B).
Because wobble base pairing could occur at any codon position and still lead to peptide bond formation before the advent of h44 ( Figure 5A, left), one should wonder how extensive miscoding could be overcome at the origin. As already suggested by F. Crick, the most plausible solution that occurred was a minimal set of anticodons and codons (41). Several lines of evidence suggest that the 'GNC' code (where N is A, G, C or U) was prevailing at the origin of translation, which would have overcome miscoding while simultaneously managing frameshifting and frame indeterminacy at that stage (42)(43)(44) (Figure 6, left).
Accuracy of translation: a GU wobble base pair is only slightly less stable than an AU base pair (45), but it is tilted compared to a regular WC base pair. In the context of the N 36 -N 1 -A 1493 triple base pair, no such degree of freedom is available due to planar constraints: in order for A1493 to establish optimal hydrogen bonds with N 36 -N 1 , this base pair has to display a WC geometry (3,4) (Table 1).
Thus, an increase in the dimensionality of the complex at the first position, which reduces the conformal freedom of the N 36 -N 1 base pair, is associated with an increased selectivity. Although extensive investigations will be required to establish how A1493 and A1492 improve the fidelity of translation without G530 and the associated inducedfit mechanism, a prevalence of mismatches at the third position should increase the overall accuracy of decoding through a higher fidelity in positions 1 and 2, required to keep the k-below a critical value enabling peptide bond formation.
It should be mentioned that Satpati et al. (46) found out through molecular dynamic simulations that mismatches on modern ribosomes are penalized essentially as a result of water exclusion due to the binding of A1493, A1492 and G530: missing hydrogen bonds occurring in mismatches cannot be compensated through hydrogen bonding with water. This effect could already partially occur without loop h18 and G530.
Another effect resulting from the action of h44 must be considered: because the A-site tRNA and the RNA template became caught by A1493 and A1492 upon anticodon binding, the rate of translocation necessarily slowed down ( Figure 4B, bottom). On modern ribosomes, the grip of the decoding center constitutes a barrier to translocation, which is overcome by the elongation factor EF-G and the free-energy available from the hydrolysis of a GTP (47)(48)(49)(50). Without helix h18 and G530, translocation could still spontaneously occur through thermal fluctuations (51). A consistent evolutionary scenario is that an ancestor of EF-G came into the picture after the emergence of A1493 and A1492, which would alleviate the early grip, and make the second transition to G530 and proofreading possible by preventing a catastrophic slowdown of translocation upon building of the full decoding center ( Figure 4B, bottom). During evolution, an early fixation of R 37 , which makes an interstrand stacking and thus helps maintain the reading frame, would also best ensure the maintenance of that frame upon appearance of A1493 and A1492 (Figures 4B and 5A). The subsequent appearance of EF-G and R 37 modifications would further reduce frameshifting events during translocation (26,27,29,50,(52)(53)(54).
Co-evolution of the translation machinery and the genetic code. This section summarizes and brings further justifications to the evolutionary model depicted in Figure 4B. Remarkably, all three major transitions highlighted in this scenario agree with the model of ribosome evolution proposed by Harish and Caetano-Anollés (19) (Figure 4A). While still being consistent with the model of Petrov et al. (20), our analysis does not support a very early appearance of the PTC on the ribosome, as suggested by this study (see summary and discussion Section).
Initial stage (0): although no strong evidence so far explains the origin of RNA and how the initial translation came about, the volume correlation in the genetic code (11) suggests that the early translation was driven by a simple Michaelis-Menten kinetic scheme (31,32,35). The fixation of U 33 and R 37 on the anticodon loops, which improved anticodon-codon associations and helped maintain the reading frame (26,29), was plausibly an early acquisition on all tRNAs.
First major transition (1): residues A1493 and A1492 appeared on helix h44. Together with U 33 and R 37 , they established the basis of modern degeneracy (Figures 5 and 6, center).
Second major transition (2): build-up of the PTC. Because this catalytic site confines the aminoacyls in a desolvated environment, the amino groups are more reactive (55). Furthermore, an induced-fit mechanism orients the aminoacyls for nucleophilic attack, which cancels the conformational freedom available to the amino group in solution, that is side-chain dependent (35). As a consequence, all k pep s are levelled up to an approximately uniform k pep value.
Nucleic Acids Research, 2022, Vol. 50, No. 7 4121 A B strengthening Figure 5. Evolutionary transition 1: from early tRNA anticodon loops and no decoding center to U 33 and R 37 -shaped anticodon loops and helix h44 on the ribosome (A1493 and A1492). (A) Left: initial loop of tRNA adapter, with little structuration, bound to a codon in an absence of decoding center. Although the shape of the loop might provide a high flexibility to the base pair at the third position, single GU wobble base pairs may also occur in pos. 2 or 1 (background) while still providing enough stability to ensure peptide bond formation in the early translation mechanism. Right: the advent of R 37 and helix h44 strengthened the anticodon-codon interaction at the first position, while the U-turn (U 33 ) helped relax base pairing specificity at the third position. R 37 stacking on N 36 -N 1 is schematized with a thin red line. (B) Translation of early coding sequences: suggested improved processivity resulting from transition 1. Because the early replication mechanism is inaccurate, RNA sequences accumulate mutations, and thus may not always be fully translated due to reduced sets of tRNAs (left). The advent of h44 together with anticodon loop structuration (see A) provided an improved processivity during translation by lowering base pairing requirement at the third position (right).   . Anticodon-codon interaction and codon degeneracy in the genetic code during ribosome evolution. From an early hypothetical structure with no decoding center (initial state, 0), in which the properties of a GNC code may have provided a required stability to an early translation system (42,44), evolutionary models and the dynamics of the decoding center suggest that helix h44 with A1493 and A1492 appeared first (transition 1), which enabled an extended degeneracy at the third position (blue boxes). The completion of the PTC (transition 2) and the appearance of EF-Tu (proofreading) necessarily occurred before a controlled hydrolysis on EF-Tu by the decoding center through G530 and 30S closure (transition 3), which gave rise to modern degeneracy. Inferred kinetic scheme and codons occurring from stage 0 to transition 3 are indicated at the bottom.
Thus, at the time of the completion of the PTC, the optimization in rate/decoding that had guided the establishment of the code became obsolete. Free from this constraint, the genetic code could evolve on its own, although codon reassignment is known to have occurred at an extremely low rate--otherwise, the volume correlation would have disappeared.
Because it would break the initial simple MM kinetic scheme ( Figure 4B, left), the EF-Tu cofactor could come into the picture only after the optimization of the k' pep achieved by the PTC. In the absence of G530 and inducedfit mechanism, an elementary form of proofreading would occur: most plausibly, GTP hydrolysis on EF-Tu, that leads to the release of the tRNA for accommodation (30), was initially triggered by the docking of the tRNA•EF-Tu•GTP ternary complex onto the saricin-ricin loop of the ribosome, which would occur without requiring the kick by the 30S, thus following the simple clockwork mechanism envisioned by Ninio and Hopfield (21)(22)(23), which is independent of the decoding center.
Third major transition (3): appearance of helix h18 and the associated induced-fit mechanism (56), that involves G530 anticodon-codon latching and 30S closure (3,4,14,15,18). This large-scale rearrangement docks EF-Tu on the saricin-ricin loop, which triggers GTP hydrolysis (15,16). From the early simple proofreading mechanism (see above), a plausible evolutionary transition was a change in the structure of the ribosome architecture that kept the tRNA•EF-Tu•GTP ternary complex away from the saricin-ricin loop, thus requiring 30S closure to dock the complex.
Available data (55,57) suggest that the kinetic constant of accommodation (k acc ) is of the same order of magnitude as k' pep at physiological pH on modern ribosomes ( Figure 4B, right), although this point still needs to be established experimentally. Because of its sensitivity, that is tuned by tRNA deformation (17,38), the induced fit would allow a much sharper discrimination between cognate and near-cognate tRNA through optimal decoding (36,37), thus giving rise to modern degeneracy (Figure 6, right). Base modifications, which could only occur at a late stage with modifying enzymes, will still be required to shape some tRNA anticodon loops so that they can be accepted by the decoding center (58,59), best prevent leaking wobbling between contiguous two-fold degenerate codon families, and ensure reading frame maintenance during translocation.
According to this evolutionary model, the key roles of A1493 and A1492 in degeneracy--i.e. when N 36 -N 1 and N 35 -N 2 are both WC--were 'shifted' from the step just prior to peptide bond formation (at the origin) to the kinetic step of GTPase activation on EF-Tu (on modern ribosomes). At the origin, A1493 and A1492 binding compensated mismatches at the third position by keeping k-under a critical value enabling peptide bond formation. In the kinetic step of GTPase activation on modern ribosomes, A1493 still has a compensatory role for unspecific N 34 -N 3 base pairs through N 35 -N 2 stabilization on bent tRNAs, while A1492 and G530 constitute a switch monitoring the WC geometry of N 35 -N 2 . Because of the sensitivity of the switch, half of the 16 N 1 N 2 codon doublets, for which the stability of N 35 -N 2 is near the threshold for A1492 and G530 binding, can be differentiated into two neighboring two-fold degeneracy families. The higher stability of N 35 -N 2 of the 8 other N 1 N 2 codon doublets keeps them away from this threshold with any U 34 -N 3 base pair, implying that these doublets are all four-fold degenerate (see Figure 3).

SUMMARY AND DISCUSSION
Recent cryo-EM structures have revealed the dynamics of the decoding center of the ribosome during tRNA selection (15,18). Based on these results, the present work shows that residue A1493 of the decoding center plays a key role in degeneracy by strenghtening the N 36 -N 1 base pair during tRNA sampling, which allows non-specific N 34 -N 3 pairings to be accepted by the ribosome. This possibility was suspected at the time of an earlier work on degeneracy (12), although it remained unclear because the dynamics of the decoding center was unknown. We now conclude that degeneracy in the modern genetic code is established by a complex comprising the anticodon, the codon and A1493, while a clear-cut distinction between contiguous two-fold degenerate families requires the induced fit mediated by the whole decoding center and modifications on tRNA anticodon loops.
It must be emphasized that degeneracy corresponds to a maximization of wobbling (12), which requires specific tRNAs. Decoding in mitochondria suggests that a uridine in position 34 can almost always achieve superwobbling in four-fold degenerate families (6,7,9), while some uridine modifications, such as uridine 5-oxyacetic acid, are known to further enhance this property (60,61).
However, most bacteria and higher order organisms use more than one tRNA to translate all codons in four-fold degenerate codon families. Although a general structural strategy that prevents superwobbling has not been identified, the absence of this flexibility results from constraints applied to the anticodon-codon interaction, which could occur either through U 34 modifications, by the choice of the anticodon 32-38 closing base pairs (62) or through the flexibility of the whole tRNA (39).
Furthermore, in twofold degenerate codon families, U 34 modifications (e.g. xm 5 s 2 U derivatives) are always present, and are required to prevent 'leaking' wobbling between families sharing identical nucleotides in positions 1 and 2 (63,64). Codon assignment was, therefore, partially ambiguous before the appearance of modifying enzymes (and still is to some extent). The advent of inosine might explain why AUR and AUY two-fold degenerate families further reorganized into AUG and AU/U,C,A codon boxes. More generally, the extent of wobbling--and thus, degeneracy--is controlled by structural deformations required for the anticodon to achieve proper codon binding in the context specified by the ribosome and EF-Tu (17,(36)(37)(38)(39), which often requires base modifications (58,59,65).
The reason why degeneracy has remained as the full extension of wobbling is likely that the tRNAs of the earliest simple organisms (perhaps LUCA) pushed the limits of wobbling to the maximum. Once these rules were established and encoded in genomes, it became (almost) impossible to change them although more tRNAs following simpler (or no) wobbling rules were added afterwards, which offered more flexibility and efficiency in decoding.
According to the present analysis, a maximum of 24 degeneracy families may occur--neglecting a few singularities such as that of AUG and AU/U,C,A codon boxes in the canonical genetic code, implying that 23 amino acids may be encoded if only one degeneracy family is assigned to stop codons. In view of the interest in genetic code expansion technology (66)(67)(68), one should wonder if there are physical limits to the maximum number of amino acids that may be unambiguously translated (setting aside the issue of aminoacylation). It should first be acknowledged that >30 different tRNAs are still required in Escherichia coli for the translation of all its coding genes because superwobbling does not occur in this bacterial species, although tRNAs with inosine in position 34 can translate U/C/A-ending codons, while other modifications such as 5-hydroxyuridine are known to confer additional wobbling capabilities. It implies that it should in principle be possible to encode >30 amino acids with the existing translation machinery. It is still very hypothetical since no existing organism could tolerate massive codon reassignment.
The structural analysis of degeneracy suggests yet another theoretical possibility: if instead of G and C, another set of natural or synthetic nucleotides orthogonal to A and U and pairing with only two hydrogen bonds is used, only two-fold degenerate codon families might occur in the entire table as a result of the low stability of all N 35 -N 2 base pair. This would allow the unambiguous encoding of 31 amino acids plus one degeneracy family assigned to stop codons.
Although it is unclear whether a set of bases pairing with two hydrogen bonds could have occur at the origin of Life in addition to A and U, no k-≈ k pep optimization is possible with only weak base pairs, which may contribute to explain why this scenario did not occur.
While the present analysis shows that hydrogen bonds determine the extent of degeneracy in the genetic code, experiments and molecular dynamic simulations suggest that steric complementarity between the decoding center and the anticodon-codon complex is more important than hydrogen bonds in the selection of cognate tRNAs (69,70). There is, however, no fundamental contradiction between these two results: the network of hydrogen bonds involved in degeneracy contributes to the stabilization of the WC geometry at the second position, which is critical only when a non-canonical base pair occurs at the third position. The expected effect of missing hydrogen bonds is only a reduction of the extent of wobble base pairs accepted by the ribosome: in particular, superwobbling with U 34 that normally occur would be prohibited when specific hydrogen bonds are missing.
In the evolutionary scenario depicted in Figure 4B, the PTC emerges after helix h44 and before helix h18, in agreement with the analysis of Harish and Caetano-Anollés (19) ( Figure 4A). This succession can be justified by the following: because the PTC levelled the kinetic constants of peptide bond formation up to similar k' pep values, it cancelled the k-≈ k pep adjustment that had shaped the code from the origin (11,31,35). This early optimization, the trace of which is the volume correlation (11), could not have occurred if the PTC was already present at the origin of translation. According to the present work, the possibility of the A1493(h44)/degeneracy rebalancing is based on this optimization, implying that h44 necessarily emerged before the PTC. Our results thus do not support an early emergence of this catalytic site, as the model of Petrov et al. (20) suggests. Another justification of the proposed evolutionary scheme relates to tRNA accommodation, which is part of the proofreading mechanism, and implies the presence of the PTC: the tRNA acceptor arm is funnelled by rRNA helices H89 and H90-92, which are both rooted on this catalytic site (71,72). Also, proofreading implies a commitment of the ribosome to peptide bond formation once the 3' end of an aminoacyl-tRNA reaches the peptidyl-tRNA, which implies high k' pep s of similar values, thus the PTC. We conclude that in the timeline of evolution, the completion of the PTC occurred after the appearance of degeneracy (residues A1493 and A1492) and before EF-Tu/proofreading, the induced-fit mechanism (30S closure) controlled by G530 being necessarily a latecomer.
One of the most striking structural aspect of the decoding center is that its three nucleotides are distributed on two different helices far apart from each other, implying that their simultaneous appearance in the course of the early evolution of the ribosome is highly unlikely. In agreement with evolutionary models (19,20), and with the dynamics of the decoding center (15,18), the major conclusion of the present analysis is that degeneracy arose when helix h44 together with residues A1493 and A1492 appeared at an early stage of the evolution of the ribosome.